1. Field of the Invention
The present invention relates generally to methods for culturing and genetically altering higher plants. More particularly, the present invention relates to methods for culturing and genetically transforming tissue material from carnation plants.
The carnation, Dianthus caryophyllus, is a popular ornamental plant and highly valued for its cut flowers. As with many ornamental plant species, breeders have long sought to improve existing varieties and to create new cultivars using conventional techniques, such as cross-breeding and somatic clonal variation. Phenotypic variations of particular interest include color, fragrance, morphology, herbicide resistance, pesticide resistance, environmental tolerance, vase life of the cut flower, and the like. While improvements and variations in many or all of these characteristics have been achieved, progress is slow because of the inherently random nature of such breeding approaches. Indeed, the introduction of any particular characteristic requires a substantial effort if it can be achieved at all.
The propagation of carnation plants through the regeneration of plant parts and callus, as well as through micropropagation of shoot cultures, can be difficult even when it is not desired to introduce genetic alterations. In particular, methods proposed for the regeneration of carnation tissue often result in "vitrified" shoots having a glassy or translucent appearance and an abnormal morphology. Moreover, conventional regeneration methods often have regeneration frequencies below about 10% Micropropagation methods often are slow and produce few shoots per original cultured shoot. Those methods involving liquid medium cultures can be laborious and care subject to contamination.
For these reasons, it would be desirable to provide improved regeneration and micropropagation procedures for the efficient in vitro reproduction of carnation plant material. It would be particularly desirable to combine recombinant DNA technology with the regeneration and micropropagation techniques in order to produce new carnation cultivars in a controlled and predictable manner. Such recombinant DNA methods should provide for transformation, should be capable of introducing preselected exogenous gene(s) to the carnation plant, and should permit selection of transformed plant materials which are capable of expressing the exogenous gene(s). The method should also produce regenerated carnation plants which have stably incorporated the desired exogenous DNA sequences.
2. Description of the Background Art
Woodson (1989) Hort. Science 24:80 (Abstract No. 172) briefly describes the use of Agrobacterium tumefaciens to transform carnation petal explants. While the regeneration of roots in putative transformants is asserted, no regeneration of whole plants is described. The regeneration of carnation plants (without transformation) from tissue has been described. See, e.g., Petru et al. (1974) Biologia Plantarum 16:450-453 (stem formation in callus from hypocotyl and apical meristems); Lesham (1986) Hort. Science 21:320-321 (regeneration from callus and petals); and Frey et al. (1989) Hort. Science 24:74 (Abstract No. 124) (regeneration from petals and other explants with a callus stage; 1.5-3% of shoots survived transfer to soil). The micropropagation (shoot multiplication) of cultured shoots and meristem material has been described. See, e.g., Hackett et al. (1987) Amer. Soc. Hort. Sci. 90:365-369 (shoot tip propagation); Earle et al. (1975) Hort. Science 10:608-610 (shoot tip propagation); Davis et al. (1977) J. Amer. Soc. Hort. Sci. 102:48-53 (shoot tip propagation); and Ziv et al. (1983) Plant Cell Tissue Organ Culture 2:55-65 (describes vitrification in shoot tip culture).